Interactions Plasmodium falciparum/Anopheles gambiae et mécanismes moléculaires de la transmission chez le vecteur

Avec environ 250 millions de cas enregistrés annuellement dans le monde, et 89% des cas recensés en Afrique sub-saharienne, le paludisme reste une des parasitoses les plus dévastatrices. Pendant le cycle de développement du parasite chez le vecteur, des pertes importantes en nombre de parasites ont été reportées; elles sont le fait du système immunitaire du moustique, qui peut éliminer complètement les parasites et interrompre ainsi la transmission de la maladie. Le séquençage du génome de l anophèle et la possibilité d invalidation des gènes par interférence d ARN (RNAi) ont permis d effectuer des analyses fonctionnelles de la réponse antiparasitaire du moustique. Dans le but d étudier le rôle de TEP1 dont l activité contre P. berghei a été élucidée par RNAi, dans le contexte naturel, les moustiques invalidés pour TEP1 ont été gorgés sur membrane à partir du sang des porteurs de gamétocytes de P. falciparum. L utilisation de sept marqueurs microsatellites pour le génotypage des gamétocytes, a révélé que la MOI est un paramètre primordial qui prédéfinit le phénotype d un gène candidat; TEP1 n exerçant son activité antiparasitaire que dans les cas d infections de MOI 2. L étude comparative de la réponse immune d A. gambiae vis-à-vis des parasites humain et de rongeurs, a révélé une sensibilité distincte de chacune des espèces parasitaire à une arme de la réponse immune du moustique. Nous avons par ailleurs identifié deux nouveaux acteurs de cette réponse, TGase9098 et Kay, spécifiquement impliqués dans la défense du moustique contre l infection à P. falciparum. A partir de la mesure de l expression des peptides antimicrobiens Defensine1, Gambicine et TEP1 par PCR quantitative, nous avons mis en exergue le fait que cette immunité basale puisse avoir un impact sur la compétence vectorielle des stades adultes.Les différences phénotypiques observées entre les parasites, clonal de rongeurs, et les isolats naturels de P. falciparum, confirment la complexité des interactions rencontrées en condition naturelle de transmission. L identification des acteurs antiparasitaires spécifiquement dirigés contre P. falciparum, constitue un atout majeur dans la mise en place ainsi que dans l amélioration des mesures de lutte visant à bloquer le développement parasitaire chez son vecteur naturel.With 250 million of clinical cases annually, of which 89% occur in sub-Saharan Africa, malaria remains one of the most devastating infectious disease in the world. During its life cycle within a mosquito, the parasite undergoes dramatic losses attributed to mosquito immunity, which in some cases can efficiently block parasite development leading to total mosquito refractoriness to Plasmodium. The genome sequencing of A. gambiae and the development of reverse genetic tools such as in vivo gene silencing using dsRNA, benefited dramatically functional analysis of mosquito antiparasitic responses.During my PhD studies, I investigated the role of TEP1, the first mosquito factor identified that mediates killing of P. berghei ookinetes, using experimental infections with fields isolates of P. falciparum in Cameroon. I have demonstrated that TEP1 mediates killing of the human malaria P. falciparum but, surprisingly, the antiparasitic effect of TEP1 was detected only in monoclonal infections or infections with low genetic complexity (MOI <= 2). These studies revealed a new parameter that regulates outcome of infections in natural conditions, which should be taken into consideration for accurate result analysis. Further, our comparative analysis revealed that rodent and human parasites were sensitive to distinct arms of the mosquito immune responses. I demonstrated that P. falciparum parasites were particularly sensitive to mosquito wounding-induced responses and identified two new components of these responses, AP1/Kay and TGase9098.I have then tested a hypothesis that mosquito basal immunity induced at larval stages impacted vector competence of mosquitoes. Transcriptional analysis of expression of antimicrobial peptides defensin 1, gambicine and TEP1 in A. gambiae larvae collected in a series of larval breeding sites in Cameroon, suggested that higher levels of expression of immune markers correlated with lower Plasmodium infection rates in adults. The identification of factors that limit P. falciparum development should provide new approaches for innovative control measures to block parasite development within the mosquito.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Dynamics of bacterial community composition in the malaria mosquito's epithelia

The Anopheles midgut hosts diverse bacterial communities and represents a complex ecosystem. Several evidences indicate that mosquito midgut microbiota interferes with malaria parasite transmission. However, the bacterial composition of salivary glands and ovaries, two other biologically important tissues, has not been described so far. In this study, we investigated the dynamics of the bacterial communities in the mosquito tissues from emerging mosquitoes until 8 days after a blood meal containing Plasmodium falciparum gametocytes and described the temporal colonization of the mosquito epithelia. Bacterial communities were identified in the midgut, ovaries, and salivary glands of individual mosquitoes using pyrosequencing of the 16S rRNA gene. We found that the mosquito epithelia share a core microbiota, but some bacteria taxa were more associated with one or another tissue at a particular time point. The bacterial composition in the tissues of emerging mosquitoes varied according to the breeding site, indicating that some bacteria are acquired from the environment. Our results revealed temporal variations in the bacterial community structure, possibly as a result of the mosquito physiological changes. The abundance of Serratia significantly correlated with P. falciparum infection both in the midgut and salivary glands of malaria challenged mosquitoes, which suggests that interactions occur between microbes and parasites. These bacteria may represent promising targets for vector control strategies. Overall, this study points out the importance of characterizing bacterial communities in malaria mosquito vectors

K-13 propeller gene polymorphisms isolated between 2014 and 2017 from Cameroonian Plasmodium falciparum malaria patients.

The emergence of artemisinin-resistant parasites since the late 2000s at the border of Cambodia and Thailand poses serious threats to malaria control globally, particularly in Africa which bears the highest malaria transmission burden. This study aimed to obtain reliable data on the current state of the kelch13 molecular marker for artemisinin resistance in Plasmodium falciparum in Cameroon. DNA was extracted from the dried blood spots collected from epidemiologically distinct endemic areas in the Center, Littoral and North regions of Cameroon. Nested PCR products from the Kelch13-propeller gene were sequenced and analyzed on an ABI 3730XL automatic sequencer. Of 219 dried blood spots, 175 were sequenced successfully. We identified six K13 mutations in 2.9% (5/175) of samples, including 2 non-synonymous, the V589I allele had been reported in Africa already and one new allele E612K had not been reported yet. These two non-synonymous mutations were uniquely found in parasites from the Littoral region. One sample showed two synonymous mutations within the kelch13 gene. We also observed two infected samples with mixed K13 mutant and K13 wild-type infection. Taken together, our data suggested the circulation of the non-synonymous K13 mutations in Cameroon. Albeit no mutations known to be associated with parasite clearance delays in the study population, there is need for continuous surveillance for earlier detection of resistance as long as ACTs are used and scaled up in the community

Preliminary validation of the use of IgG antibody response to Anopheles gSG6-p1 salivary peptide to assess human exposure to malaria vector bites in two endemic areas of Cameroon in Central Africa.

The specific immune response to the Anopheles salivary peptide could be a pertinent and complementary tool to assess the risk of malaria transmission and the effectiveness of vector control strategies. This study aimed to obtain first reliable data on the current state of the Anopheles gSG6-P1 biomarker for assess the level of exposure to Anopheles bites in high malaria endemic areas in Cameroon. Blood smears were collected from people living in the neighborhoods of Youpwe (suburban area, continental) and Manoka (rural area, Island), both areas in the coastal region of Cameroon. Malaria infection was determined using thick blood smear microscopy, whereas the level of specific IgG response to gSG-P1 peptide was assessed by enzyme-linked immunosorbent assay from the dried blood spots. Of 266 (153 from Youpwe, 113 from Manoka) malaria endemic residents (mean age: 22.8±19.8 years, age range: 6 months-94 years, male/female sex ratio: 1/1.2, with Manoka mean age: 23.71±20.53, male/female sex ratio:1/1.13 and Youpwe mean age: 22.12±19.22, male/female sex ratio 1/0.67) randomly included in the study, Plasmodium infection prevalence was significantly higher in Manoka than in Youpwe (64.6% vs 12,4%, p = 0.0001). The anti-gSG6-P1 IgG response showed a high inter-individual heterogeneity and was significantly higher among individuals from Manoka than those from Youpwe (p = 0.023). Malaria infected individuals presented a higher anti-gSG6-P1 IgG antibody response than non-infected (p = 0.0004). No significant difference in the level of specific IgG response to gSG-P1 was observed according to long lasting insecticidal nets use. Taken together, the data revealed that human IgG antibody response to Anopheles gSG-P1 salivary peptide could be also used to assess human exposure to malaria vectors in Central African region. This finding strengthens the relevance of this candidate biomarker to be used for measuring human exposure to malaria vectors worldwide

Effectiveness of Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine in Pregnancy: Low Coverage and High Prevalence of <i>Plasmodium falciparum</i> dhfr-dhps Quintuple Mutants as Major Challenges in Douala, an Urban Setting in Cameroon

Intermittent preventive treatment in pregnancy with sulfadoxine and pyrimethamine (IPTp-SP) is a key component in the malaria control strategy implemented in Africa. The aim of this study was to determine IPTp-SP adherence and coverage, and the impact on maternal infection and birth outcomes in the context of widespread SP resistance in the city of Douala, Cameroon. Clinical and demographic information were documented among 888 pregnant women attending 3 health facilities, from the antenatal care visit to delivery. Positive samples were genotyped for P. falciparum gene (dhfr, dhps, and k13) mutations. The overall IPTp-SP coverage (≥three doses) was 17.5%, and 5.1% received no dose. P. falciparum prevalence was 16%, with a predominance of submicroscopic infections (89.3%). Malaria infection was significantly associated with locality and history of malaria, and it was reduced among women using indoor residual spraying. Optimal doses of IPTp-SP were significantly associated with reduced infection among newborns and women (secundiparous and multiparous), but there was no impact of IPTp-SP on the newborn bodyweight. Pfdhfr-Pfdhps quintuple mutants were over-represented (IRNI-FGKAA, IRNI-AGKAA), and sextuple mutants (IRNI-AGKAS, IRNI-FGEAA, IRNI-AGKGS) were also reported. The Pfk13 gene mutations associated with artemisinin resistance were not detected. This study highlights the role of ANC in achieving optimal SP coverage in pregnant women, the mitigated impact of IPTp-SP on malaria outcomes, and the high prevalence of multiple SP-resistant P. falciparum parasites in the city of Douala that could compromise the efficacy of IPTp-SP

Antimalarial drug resistance in the Central and Adamawa regions of Cameroon: Prevalence of mutations in P. falciparum crt, Pfmdr1, Pfdhfr and Pfdhps genes

International audienceThe spread of Plasmodium falciparum resistant parasites remains one of the major challenges for malaria control and elimination in Sub Saharan Africa. Monitoring of molecular markers conferring resistance to different antimalarials is important to track the spread of resistant parasites and to optimize the therapeutic lifespan of current drugs. This study aimed to evaluate the prevalence of known mutations in the drug resistance genes Pfcrt , Pfmdr1 , Pfdhfr and Pfdhps in two different epidemiological settings in Cameroon. Dried blood spots collected in 2018 and 2019 from asymptomatic individuals were used for DNA extraction and then the Plasmodium infection status was determined byPCR. Detection of SNPs was performed by nested PCR followed by allele-specific restriction analysis (ASRA). The prevalence of each genotype was compared between sites using the Chi square and Fisher’s exact tests. A high prevalence of the Pfcrt K76 wild type allele was found in both sites (88.5 and 62.29% respectively; P 96%), however no SNP was detected at codon 164. In Pfdhps , the prevalence of the 437G mutation reached (90%) and was at higher frequency in Mfou (P< 0.0001). Overall, the Pfdhps mutations 540E and 581G were less common (0.33 and 3.26%, respectively). The quadruple resistant genotype ( Pfdhfr 51I/59R/108N+ Pfdhp 437G) was found almost 90% of the samples. The wild-type genotype ( Pfdhfr N51/C59/S108/164I+ Pfdhps A437/K540/A581) was never identified and the sextuple mutant ( Pfdhfr 51I/59R/108N+ Pfdhp 437G/540E/581G), kwon as super resistant appeared in two samples from Tibati. These findings demonstrate declining trends in the prevalence of mutations conferring resistance to 4-aminoquinolines, especially to chloroquine. However, a high level of mutations in P . falciparum genes related to SP resistance was detected and this raises concerns about the future efficacy of IPTp-SP and SMC in Cameroon

High Prevalence of Polyclonal <i>Plasmodium falciparum</i> Infections and Association with Poor IgG Antibody Responses in a Hyper-Endemic Area in Cameroon

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p p P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies